Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

Protective and Therapeutic Effects of an IL-15:IL-15Rα-Secreting Cell-Based Cancer Vaccine Using a Baculovirus System

This study reports the use of the BacMam system to deliver and express self-assembling IL-15 and IL-15Rα genes to murine B16F10 melanoma and CT26 colon cancer cells. BacMam-based IL-15 and IL-15Rα were well-expressed and assembled to form the biologically functional IL-15:IL-15Rα complex. Immunization with this IL-15:IL-15Rα cancer vaccine delayed tumor growth in mice by inducing effector memory CD4+ and CD8+ cells and effector NK cells which are tumor-infiltrating. It caused strong antitumor immune responses of CD8+ effector cells in a tumor-antigen specific manner both in vitro and in vivo and significantly attenuated Treg cells which a control virus-infected cancer vaccine could induce. Post-treatment with this cancer vaccine after a live cancer cell injection also prominently delayed the growth of the tumor. Collectively, we demonstrate a vaccine platform consisting of BacMam virus-infected B16F10 or CT26 cancer cells that secrete IL-15:IL-15Rα. This study is the first demonstration of a functionally competent soluble IL-15:IL-15Rα complex-related cancer vaccine using a baculovirus system and advocates that the BacMam system can be used as a secure and rapid method of producing a protective and therapeutic cancer vaccine.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA)

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Progress in the development of vaccines and strategies of their application in the control of infectious diseases of swine

Data are presented concerning vaccines used in the prophylaxis and control of some viral and bacterial infectious diseases of swine, particularly against: MPS, PRRS, PMWS CSF, AD, SI, E. coli, Cl. perfringens type C and A and oedema disease. The lower efficacy in immunization of inactivated vaccines is stressed. However in case of live vaccines against PRRS this is not always the case since also live attenuated vaccines deliver unsatisfactory results of immunization. This is explained by frequently occurring genetic changes in the genome of the field PRRSV strains in comparison with the strain or strains being in the vaccine. Vaccines against PMWS are characterized, underlining high efficacy of the vaccines in the Baculovirus system. After withdrawing from field use against CSF the vaccines Lapest and Cellpest a recombinant vaccine against CSF was introduced for control of this disease. A recombinant vaccine against AD of swine is mentioned, which was successfully used in eradication program in Europe. Of high practical value is mentioned a vaccine against SI containing subptypes H1N1, H3N2 and H1N1. Referring to vaccines against viral diseases of swine marker vaccines were mentioned enabling differentiation of animals infected from vaccinated (DIVA strategy). At the beginning of the XXI century several new and effective vaccines were developed, produced and used against diarrheal diseases of different etiology and for the first time against oedema disease. The role of strategy of vaccination depending on the characteristic of the infectious disease, the vaccine and the production system of swine is present.